Green fluorescence protein (hereinafter, referred to as “GFP”) was originally isolated from a jellyfish. Since GFP protein consists of a number of 238 amino acids, and since it does not need any other proteins or substrates for fluorescence activation, it has been widely used as a reporter protein. Various types of GFP mutants have been reported. EGFP (enhanced GFP) was prepared by increasing intensity of GFP's fluorescence; and BFP (blue fluorescence protein), CFP (cyan fluorescence protein) and YFP (yellow fluorescence protein) were prepared by modifying fluorescence spectrum.
In 1998, it was firstly reported that fluorescence intensity of GFP was maintained even after inserting foreign protein or a part of protein or peptides thereto at one or more sites (see NAR 26:623-630). Since then, molecules belonging to the group of the inserted fluorescence proteins, which were developed by R Tsien et al., have been used. YFPins and Camgaroo, which was made by inserting calmodulin to the YFPins, were representative inserted fluorescence proteins designed by R Tsien et al. (see PNAS 96:11241-11246). The mutant fluorescence proteins, however, did not show fluorescence activities at 37° C., while they displayed fluorescence activities at 28° C. Thus, they could not be used as biosensors in mammalian cell to measure activities of any desired materials. R Tsien et al. also reported that Camgaroo 2 (Q69M mutant), which was made by substituting 69th amino acid sequence of Glutamine with Methionin, represented fluorescence even at 37° C. However, the fluorescence intensity of the Camgaroo 2 was so weak that it could not be used in the measurement of calcium at a single cell level. Accordingly, there has been the need to find a novel inserted fluorescence protein having even stronger fluorescence.
Concerning viral disease, since the cause of disease was not defined clearly in general the obtaining of molecules, which inhibit activities of the disease-causing enzymes, has been the primary subject for the drug development these days. In this regard, an efficient cell-based assay system to monitor activities of target proteins of viral disease was required. Particularly, regarding human Hepatitis C virus, which cannot be cultivated in a laboratory, the development of a useful cell-based pharmacological assay system to define drug efficacy in a cell was greatly required. Although NS3 protease or NS5B RNA polymerase has been considered as an important target protein for the development of drug for the treatment of viral disease of human Hepatitis C, there has not been any efficient cell-based assay system to determine the effects of drug. Until now, recombinant viruses designed to have life-cycle dependent on NS3 protease activity have been used as cell-based reporting systems for detecting the activity of NS3 protease. Jang, seung-gi reported in 1996 an assay system using NS3 protease dependent poliovirus (see Virology 226:318-26), and Jecyca et al. reported in 1998 other assay system using Sindbis virus for detecting NS3 protease activity (J. Virol 73:561-575). In addition, other assay system using BVDV (bovine viral diarrhea virus) was disclosed in a published document in 2000 (see J. Virol 74: 6339-6347). These assay systems for detecting and analyzing NS3 protease's activity using viruses belonging to Flaviviridae family, which had similar molecular biological features, were proved to have useful features. However, these systems had several problems to be resolved as high through put assay systems. These systems regarded to use original viruses as controls, and viral infection problem arose after the cell culture, in the procedures of detecting inhibitors. Therefore, the demand for more efficient and more cost effective assay system is still very high.
It is known that caspase recognizes and cleavages a protein at the site of amino acids following aspartic acid. In 1998, Xu et al. detected caspase-3 (CPP32) activity using FRET (Fluorescence Resonance Energy Transfer) that was caused by placing DEVD (SEQ ID No.: 17) amino acid sequence between GFP and BFP (see NAR 26:2034-2035). Also, BD bioscience clontech designed a system to monitor the activity of caspase-3 through tracing and investigating the YEP within a cell by fusing DEVD-YEP and nuclear export sequence (BD bioscience clontech, PR1Z499W). However, in detecting caspase activity using FRET, signal/noise (S/N) ratio was too low for practical application in the assay system. In addition, this assay system, which was basically based on protein movement in a cell, required relatively expensive device, and it was difficult to digitize the enzyme activity since the detected results were secondary signals. Accordingly, there have been great needs to find more efficient and cost effective cell-based assay system to detect and analyze the activities of materials.
Thus, in order to provide biosensors for studying activities of numerous desired materials in cells, we carried out researches to develop new type of inserted fluorescence proteins maintaining appropriate fluorescence intensities around 37° C., and thereby we designed enhanced inserted yellow fluorescence proteins by inducing mutations to the inserted fluorescence proteins. Then, we designed a new type of biosensor for detecting activity of NS3 protein inhibitor of human Hepatitis C virus by inserting the NS3 protein inhibitor's substrate recognition sites into the enhanced inserted fluorescence protein. Also, we designed a new type of calcium sensor for determining the amount of calcium in a cell by inserting calmodulin recognition sites into the prepared enhanced inserted fluorescence protein. Furthermore, we developed a new type of caspase sensor to detect caspase activity in a cell by inserting caspase recognition site into the obtained enhanced inserted fluorescence protein.